Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/12—Well or multiwell plates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/04—Flat or tray type, drawers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/06—Plates; Walls; Drawers; Multilayer plates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/10—Perfusion

Definitions

• the present invention is in the field of perfusion culture of cells.
• the invention apparatus and method involve a bioreactor which allows for flow-through of media while retaining non-adherent as well as adherent cells within the bioreactor chamber.
• the invention is especially suitable for the culture of hematopoietic cells.
• Cell types which adhere to the surface of a culture flask may have their media exchanged or refreshed by simply pouring off the spent media and pouring in fresh media. Alternatively, a portion of the spent media may be gently drawn off and replaced with fresh media. Perfusion or flow-through of fresh media may be desirable for the growth of adherent cell types which require frequent or constant refreshment of culture media.
• adherent cells may be adversely affected by the shear stress inflicted by the bulk flow of media.
• Adherent cells may be forced away from their moorings by the bulk media flow, and then lost from the culture system. Alternatively, adherent cells may stay attached to their substrate, but be adversely affected by the force of the fluid such that they fail to proliferate and/or differentiate. Part of the adverse effects of perfusion cultures may be attributed to dilution and wash out of factors produced by the cells themselves, when those factors are necessary for cell development.
• Non-adherent cells may be retained in bioreactors with the use of physical barriers.
• a physical barrier may be in the form of a membrane that creates a barrier to the passage of cells, but allows the diffusion of nutrients and metabolic byproducts.
• Hollow fiber bioreactors work on the principle of physical barriers.
• the cells are retained behind a semi-permeable membrane (i.e., the fiber material) .
• a typical hollow fiber unit contains thousands of individual hollow fibers.
• the cells are cultured in the spaces surrounding the fibers. Culture media is perfused through the spaces, and metabolic byproducts diffuse through the semi-permeable membrane, into the hollow fibers, and then out of the system.
• Examples of hollow fiber bioreactors are disclosed in WO 91/18972 (Knazek) and WO 92/10564 (Culver) .
• bioreactors are based on the use of semi- permeable membranes or supports (U.S. 5,264,344 (Sneath) and U.S. 5,223,428 (Rose).
• roller-bottle type of bioreactor is designed for even distribution of medium throughout the cell population.
• cells adhere to the inner surface of the bottle, which is constantly rotated to bathe the cells.
• Certain roller-bottle bioreactors have increased inner surface area provided by support strips or corrugations (U.S. 5,010,013 (Serkes) ; EP 345 415 (Tyndorf) ; U.S. 3,853,712; U.S. 5,270,205 (Rogalsky) ; U.S. 5,256,570 (Clyde) ) .
• stirred bioreactors Other types of bioreactors, known as stirred bioreactors, often include the use of spin-filters and settling tubes in order to retain cells (U.S. 4,760,028 (deBruyne) ; U.S. 4,906,577 (Armstrong)). Anchorage-dependent cells may be grown on microcarrier beads, which are commonly used in stirred bioreactors (EP 046,681 (Tolbert); U.S. 5,002,890 (Morrison) ) .
• U.S. 4,939,151 discloses a cell culture bag having a non-smooth inner surface to prevent the inner surfaces from sticking together during manufacturing and sterilization processes.
• a three- dimensional solid matrix has also been proposed for growing adherent cells (US 4,514,499 (Noll).
• Hematopoietic cells are obtained from a donor's or a patient's bone marrow or peripheral blood.
• the starting cell suspension to be cultured may contain a variety of hematopoietic cells in various stages of differentiation.
• the cell suspension may first be subjected to certain selection processes, resulting in a starting cell sample highly enriched for stem cells, for instance.
• Stem cells are primitive hematopoietic cells which have the potential to differentiate into cells of all hematopoietic lineages, including granulocytes, lymphocytes, erythrocytes, and megakaryocytes.
• stem cells require adherence to a substrate in order to proliferate and develop to a progenitor stage.
• the cells that have progressed to the progenitor stage, and beyond, are thought to be generally non-adherent because their in vivo micro-environment would be a moving fluid (blood) , and they would not be adapted for adherence to a static surface.
• a culture of hematopoietic cells may contain a variety of different cell types including adherent and non-adherent cells. To further complicate the picture, some of the non-adherent cells may adhere to other cells which, in turn, adhere to a surface.
• He atopoeitic cells present additional challenges because they are shear sensitive. Hematopoietic cells do not appear to grow well when suspended in spinner flask cultures. In attempts to provide a micro-environment conducive to hematopoietic cell growth, growth surfaces have been provided with stromal layers.
• the stromal layer is generally selected to mimic the extracellular matrix in the bone marrow and consists of proteins such as collagen and fibronectin.
• Bioreactors which depend on the use of stroma are disclosed in WO 90/15877 (Emerson) , WO 92/11355 (Emerson), EP 0 358 506 (Naughton) , US 5,160,490 (Naughton) , and US 4,963,489 (Naughton) .
• stroma is disadvantageous for several reasons. First, it is time consuming to produce the stromal layer on a cell culture surface, and great care must be taken not to introduce contaminants into the culture vessels. Certain techniques for laying down stroma require the use of living cells, such as fibroblasts, which are different from the cell type to be cultured. The introduction of foreign cell types into a culture vessel complicates the task of culturing a hematopoietic cell suspension suitable for clinical use.
• a primary object for this invention is to provide a bioreactor which allows for the exchange of media without undue perturbation or loss of the cultured cells.
• Another object for this invention is to provide a bioreactor which permits retention of cells without the use of stroma.
• Another object of this invention is to provide a flow- through bioreactor which permits cultured cells to be easily and efficiently recovered from the bioreactor chamber.
• a further object of this invention is to provide a method for the perfusion culture of hematopoietic mononuclear cells, unselected for CD34+.
• Figure 1 provides a partially schematic front quarter perspective view of a flow-through bioreactor with grooves for cell retention, according to the present invention.
• Figure 2 provides a longitudinal cross-sectional view of the bioreactor of the invention.
• Figure 3 provides a cross-sectional view through the inlet port and along the length dimension of a groove.
• Figure 4 provides enlarged fragmentary cross-sectional views of the grooves.
• the bioreactor vessel 10 is shown with the lid 12 expanded from the view of the receptacle 14, in order to show the details of the inner surface of bottom wall 16.
• the lid 12 is sealed to the receptacle 14 by means known in the present art.
• the lid 12 may be permanently sealed to the receptacle 14 by means of chemical bonds, or may be sealed by means of a gasket and clamp.
• the entire bioreactor vessel 10 may be molded in one piece.
• the bioreactor vessel 10 is made of a clear plastic material such as polycarbonate, polysulfonate, acrylic, or polystyrene.
• the inner surface of the vessel 10 may also be coated with teflon or another polymer, or may have a negative charge added, according to the growth requirements of the particular cell type to be cultured.
• bottom wall 16 is provided with a plurality of long rectangular grooves 18 in which cells are retained while culture medium flows along the longitudinal axis L of the receptacle 14, in a direction transverse to the length dimension X of the grooves 18. Grooves 18 are disproportionately enlarged in this figure for better illustration.
• the lid 12 has an inlet port 20, for conveying liquid media through inlet slot 22.
• the media flows from inlet slot 22, along the longitudinal axis L through the bioreactor vessel 10, and out the outlet slot 24.
• Outlet slot 24 connects with an outlet port (26 in Figure 2) .
• the media flow is regulated by well known means such that the flow is even across the inner surface of bottom wall 16.
• means to regulate flow is provided in experimental Example 1 below.
• Figure 2 provides a longitudinal cross-sectional view of above described elements: inlet port 20, inlet slot 22, outlet slot 24, outlet port 26, inner surface of bottom wall 16, grooves 18.
• grooves 18 are disproportionately enlarged for better illustration, and groove detail has been omitted on portions of inner surface of bottom wall 16.
• grooves 18 are continuous across the inner surface of bottom wall 16.
• Inlet port 20 is connected to a reservoir of fresh media which is maintained at a suitable physiological pH by means well known in the art of cell culture.
• Outlet port 26 may be shunted to a waste container, or the media exiting outlet port 26 may be refreshed by well known means and recirculated to inlet port 20.
• Figure 3 is a cross-sectional view of bioreactor 10 in dimension X ( Figure 1) , through inlet port 20 and inlet slot 22. This sectional view runs the length of a groove 18, showing the length face 30 of a groove 18.
• Figure 4a is a cross-sectional view, perpendicular to dimension X ( Figures 1 and 3) , showing the dimensions of a groove 18 in one preferred embodiment of the invention.
• the ratio of width Y to depth Z is about 1:1.
• width Y and depth Z are each about 50 ⁇ m to about 5,000 ⁇ m.
• width Y is about 200 ⁇ m and depth Z is about 200 ⁇ m.
• groove 18 is depicted with corners and edges forming sharp 90° angles, it is understood that within the scope of this invention, corners and/or edges of the grooves might be rounded to form arcs. Given the present disclosure, it is also understood that different types of groove geometries may be devised to achieve similar results.
• Figure 4b shows the dimensions of a groove 18 in a second preferred embodiment of the invention.
• the ratio of width Y to depth Z is about 2:1.
• the preferred groove dimensions are suitable for retention of cells 32, both adherent and non-adherent, when media flows along longitudinal axis L (see Figure 1) over the inner surface of bottom wall 16, across the top of the groove 18 ( Figure 4) .
• the bulk flow of media along longitudinal axis L over the inner surface of bottom wall 16 does not perturb cells 32 within the grooves 18.
• Both adherent and non-adherent hematopoietic cells are able to proliferate and differentiate in the grooves 18 of the bioreactor of the present invention.
• a particular advantage of the method of the present invention is that a suspension of hematopoietic mononuclear cells may be successfully cultured without first selecting for CD34+ stem/progenitor cells.
• the donor's or patient's blood sample is obtained using a well-known apheresis procedure.
• the apheresis procedure may be conducted using the Baxter CS-3000TM apheresis machine, or the like. In some cases, the apheresis product is used directly without further processing.
• the apheresis product is subjected to density gradient separation to remove most red cells, platelets, and cell debris from the mononuclear cell suspension.
• the mononuclear cell suspension is placed directly into the grooved bioreactor and cultured in perfused media.
• the terms "mononuclear cells” and “mononuclear cell suspension” refer to hematopoietic cells which have been separated from most red blood cells, platelets, and multinucleated granulocytes. It is understood that the mononuclear cell suspension contains a very small fraction of CD34+ stem/progenitor cells.
• the culture method of the present invention allows the propagation and differentiation of the small number of stem/progenitor cells within the starting suspension, without disadvantageous media depletion by the numerous mature cell types in the suspension.
• Perfusion cultures in the grooved bioreactor were compared with perfusion cultures on a stromal layer (no grooves) .
• Control static cultures were performed in either a smooth surfaced flask (no grooves) or a flask with a stromal layer.
• Peripheral blood cells were obtained from two clinical sources. These cells were "mobilized” from the bone marrow of cancer patients into their peripheral blood by treatment of the patients with chemotherapeutic agents and cytokines, and collected by apheresis. The cells were received by overnight shipment in RPMI-1640 with 5% serum either on ice or at room temperature. The mononuclear cells were obtained by Ficoll density gradient (1.077 gm/cm3) centrifugation (1200 rp for 20 minutes) . The mononuclear layer obtained was washed once with 1 X Ca++ Mg++ free phosphate buffered saline (PBS) .
• PBS free phosphate buffered saline
• HLTM Human long term media
• McCoy's 5A medium supplemented with 1% MEM Vitamins, 1% 2 mM glutamine, 1% 1 mM sodium pyruvate, 1% MEM essential amino acids, 1% MEM amino acids, 1% 1M HEPES, 1% 10 mM monothioglycerol, 0.1% 50 mg/ml gentamicin sulfate (Gibco) , 12.5% preselected heat inactivated fetal bovine serum and 12.5% preselected heat inactivated horse serum.
• Colony assay medium is composed of 0.8% methylcellulose in IMDM supplemented with 50 ⁇ g/ml gentamicin sulfate, 30% preselected heat inactivated fetal bovine serum, 2% bovine albumin (Armour Pharmaceuticals) , 150 U/ml recombinant human interleukin 3 (rhIL-3, R&D Systems, Inc.), 40 ng/ml recombinant human interleukin-6(rhIL-6, Sandoz or R&D Systems, Inc.), 150 U/ml recombinant human granulocyte colony-stimulating-factor (rhG-CSF, Immunex) , 200 U/ml recombinant human granu 1 ocyte -macrophage colony-stimulating-factor (rhGM-CSF, Immunex) , and 10 U/ml recombinant human erythropoietin (rhEpo, Amgen) .
• Growth factor supplemented HLTM using the bioreactor studies contained 150 U/ml rhIL-3, 40 ng/ml rhIL-6, 150 U/ml rhG-CSF and 50 ng/ml stem cell factor (SCF, Amgen) . All of the reagents were obtained from Sigma unless otherwise specified.
• Stroma Bone marrow cultures were established as reported by Roller et al (Exp Hematol 20:264-270, 1992). Briefly, stromal cells subcultured from 2-week-old marrow cultures were used to form stromal feeder layers by inoculating into 3.75 X 7.5 cm rectangular polycarbonate dishes (Cole Par er, Chicago IL) at 4 X 10 cells/ml in 5 ml HLTM. Each dish contained a 3.75 X 7.5 Thermanox® slide (Nunc, naperville, IL) which served as the culture substratum. After a 24 hour incubation at 37°C in 5% C0 2 in air, dishes were irradiated with a dose of 12 Gy from a Cs source. The following day, cells to be cultured were seeded onto the irradiated stroma for static culture experiments. For stromal bioreactor experiments, the slides coated with stroma were rinsed and placed on the inner bottom surfaces of bioreactor vessels without grooves.
• the culture chambers were constructed of polycarbonate plastic, the tubing and connectors were constructed of Teflon, and the tubing used in the peristaltic pump was made of silicone.
• the culture chambers had the following dimensions:
• H Chamber height: 0.21 in or 0.53 cm
• Af Flow cross section (H W) 0.32 in or 2.03 cm 2
• the grooved bioreactors of the present invention also had the following dimensions:
• bioreactor All of the bioreactor parts were washed, sterilized, and reused except for the pump tubing. It is understood that, for clinical use, the bioreactor would be a single-use disposable.
• the sterile bioreactor was completely assembled in a 37°C incubator (Stericult, Forma Scientific) .
• the culture chambers were placed in a rack that kept the chambers at a uniform 10° angle from horizontal to encourage air bubbles to leave the system.
• HLTM was then circulated through the bioreactor to allow calibration of the pH and d0 2 probes. For these calibrations, the bioreactor was first equilibrated with C0 2 for the first point of the pH calibration.
• the bioreactor was equilibrated with air for the second point of the pH calibration and for the d0 2 calibration.
• the bioreactor was then drained and injected with 30 ml of HLTM and 60 ml of HLTM supplemented with 2X growth factors and the pH controller set at 7.35 ⁇ 0.05.
• the media was almost entirely drained from the three culture chambers per bioreactor prior to the seeding of the cultures.
• the cultures were seeded by injecting 10.0 ml of 2 x 10 cells/ml mononuclear cell suspension injected each of the three chambers for each bioreactor.
• the cells were allowed to settle for 15 minutes, and then the pump was started at approximately 0.2 ml/min.
• the bioreactor flow rate was measured during each feeding and the pH measured with an external pH probe (Corning) .
• Cell counts were performed on the media removed from the cultures using the Coulter Counter (Coulter Electronics) .
• One chamber per bioreactor and one corresponding control culture was harvested on days 5, 10 and 15.
• the bioreactor cultures were harvested by draining the contents, rinsing once with 10 ml of phosphate buffered saline (PBS) , rinsing once with 1 X cell dissociation solution (Sigma) , and the rinsing a second time with PBS. This was accomplished in the same manner as for the washout experiments.
• the control cultures were harvested with the same draining and rinsing schedules.
• the cell number remaining in the culture vessels was estimated by rinsing once with 10 ml of cetrimide and counting nuclei with a Coulter Counter.
• the harvested cell suspensions were concentrated by centrifugation (15 minutes at 1200 rpm) and resuspended in approximately 10 ml of fresh HLTM.
• Cell counts were performed with both a Coulter Counter and also a hemacytometer. The viability was determined by trypan blue dye exclusion during the hemacytometer counts.
• Colony assays were established at 1,000, 3,000, and 9,000 cells/ml for mononuclear cells and 500, 1,500, and 3,000 cells/ml for CD34+ cells.
• CFU-GM colony-forming-units granulocyte-macrophage
• BFU-E burst-forming-unit erythroid
• CFU-Mix mixed red and white colonies containing >50 cells were scored as colony-forming-units mixed
• LTC-IC Long-term culture initiating assays were established in 24-well tissue culture plates (Falcon) containing 1 x 10 irradiated (2,000 rad) allogeneic human bone marrow cells per well. The cells being assayed were seeded at 5 x 10 ⁇ * and 2 x 105 cells per well for the harvested mononuclear cells or 2.5 x 10 and 1 x 10 cells per well for the harvested CD34+ cells. Each well contained 2.0 ml of HLTM. The cultures are incubated at
• Stroma number of stromal cells initially seeded for stromal cultures.
• PBMN cells number of peripheral blood mononuclear cells initially seeded in both stromal and non-stromal cultures.
• Flo-Grv The flow-through grooved bioreactor of the present invention.
• Flo-Strom A flow-through bioreactor, without grooves, with a stroma-layered slide on the bottom.
• Stat/Smooth A static control culture, no stroma.
• Stat/Strom A static control culture, with stroma.
• Colony-forming units Cultures from the grooved bioreactor contained a number of granulocyte-macrophage/colony-forming units (CFU-GM) comparable to cultures from the stroma- layered bioreactor at all time points. After day 5, few erythroid cells and few BFU-E were detected in any of the cultures because the cytokine mix in the media was designed to drive granulocyte/macrophage differentiation, and not erythropoiesis.
• CFU-GM granulocyte-macrophage/colony-forming units
• Viability Cells from both types of bioreactors contained comparable number of viable cells at all time points. The viability of recovered cells was very good, ranging from 79 - 97%.
• Media supernatant analysis Media supernatant samples were analyzed for IL-6, GM-CSF, and tumor necrosis factor- ⁇ (TNF- ⁇ ) concentrations. Minimal differences were observed in cytokine concentrations between the different cultures.
• the concentration of 11-6 and TNF- increased in all cultures from about 35 ng/ml and about 25 pg/ml (day 0) to about 50 ng/ml and about 50 pg/ml (days 10-15) , respectively.
• the concentration of GM-CSF increased in the stroma-containing cultures from about 20 pg/ml (day 0) to a maximum on day 5 of about 60 pg/ml before falling to levels below input.
• the concentration of GM-CSF in the stroma-free cultures fell continuously from about 20 pg/ml (day 0) to about 5 pg/ml (days 10-15) .
• the stroma-containing cultures had a slightly faster increase in cytokine concentrations than the stroma-free cultures.
• the static cultures had a slightly faster increase in cytokine concentrations than the perfusion cultures.
• the media pH was controlled at 7.35 +/- 0.05 for the perfusion cultures, but declined from 7.35 (day 0) to about 7.25 (day 10) and about 6.90 (day 10) for the static stroma-free and stroma-containing cultures, respectively.
• EXAMPLE 2 Culture of Unselected Mononuclear Cells and CD34+ Selected Cells in the Grooved Bioreactor.
• CD34+ cells are stem cells which may require adherence to a substrate, or stroma. Therefore, it was of interest to determine whether CD34+ selected cells could proliferate in the grooves of the bioreactor of the present invention.
• stroma there is no stroma in the bioreactor of the present invention.
• the bioreactor could be formed of different types of plastics, or have plastic surfaces treated such that cells could adhere.
• the bioreactor used in the following experiments was formed of a type of plastic, polycarbonate, which is thought to be non-conducive to cell adherence since its surface is neutrally charged.
• CD34+ cells were selected from the mononuclear cell suspension by first incubating the suspension with mouse monoclonal antibodies against CD34, which bound specifically to the CD34 cell surface antigen on CD34+ cells. Then paramagnetic beads coated with sheep-anti-mouse antibodies were incubated with the cell suspension. The paramagnetic beads then bound the CD34+ cells via binding of the sheep-anti-mouse antibodies to the mouse antibodies on the CD34+ cells, to form bead/CD34+ cell complexes.
• the bead/CD34+ cell complexes were then selected from the total cell population by magnetic attraction. After washing, the CD34+ cells were released from the beads by enzymatic digestion with chymopapain. Results of CD34+ selection are shown in Table 2 below.
• the CD34+ cells were seeded into bioreactor and static control cultures as described in Example 1 above, except none of the cultures had stromal layers.
• HLTM Human long-term medium
• HLTM Human long-term medium
• IL-3 R&D Systems, Minneapolis, MN
• 40 ng/ml IL-6 Sandoz, East Hanover, NJ
• 50 ng/ml SCF Amgen, Thousand Oaks, CA
• 150 U/ml G-CSF R&D Systems
• Static cultures were performed as described in Koller M.R., et al., 1993, BioTechnol 11:358-363.
• Perfusion cultures were performed using the grooved bioreactor as described in Example 1 above.
• the perfusion culture temperature was maintained at 37.0 ⁇ 0.5°C, and the pH and dissolved oxygen (DO) data acquisition and control systems were as described in Roller, et al (supra) with the exception that the pH was controlled by a gas mixing unit with separate ports for air, N 2 , and C0 2 .
• Nonadherent cells were retained through the use of rectangular grooves, which occupied one-half of
• Perfusion and static cultures were initially seeded with either 2 x 10 mobilized peripheral blood MNCs or 2 x 10 CD34 * cells (see Example 1 above) .
• the initial cell densities were chosen to give approximately the same cell density on day 15.
• the initial medium volume was 120 ml (for 3 chambers) for perfusion cultures and 20 ml (each) for static cultures.
• the medium circulation rate in the perfusion system was gradually increased from 0 to 2.5 ml/minute/culture chamber over 1.5 hours. Negligible numbers of cells were observed in the cell trap at any time.
• Perfusion cultures were fed 3 times per week by replacing one-half of the medium with fresh HLTM and cytokines. After each chamber was harvested, the medium reservoir volume was decreased by 30 ml.
• Static cultures were fed every 5 days by replacing one-half of the medium with fresh HLTM and cytokines.
• the associated depopulation of nonadherent cells in the static cultures was 19 ⁇ 31%, as determined by cell counts on the medium removed.
• One of three parallel cultures was sacrificed every 5 days to asses total cell numbers, cell viability, CFU-GM and LTC-IC content, cell phenotype and morphology as described below. In order to prevent enzymatic damage to the cells or cell surface markers, trypsin was not used to harvest the cultures.
• Perfusion and static cultures were harvested by removing the cell suspension from the culture chamber or petri dish, rinsing with 10 ml of Ca++ and Mg++ free phosphate buffered saline (CMF-PBS, Gibco, Grand Island, NY) , rinsing with 10 ml cell dissociation solution (Sigma, #C-5789) , and rinsing a second time with 10 ml CMF-PBS. The cells were then washed and resuspended in HLTM. Cell counts and viability were determined using a hemacytometer with trypan blue dye exclusion.
• CMF-PBS Ca++ and Mg++ free phosphate buffered saline
• the nonenzymatic harvest procedure recovered greater than 97% of total cells, as determined by rinsing the harvested culture chamber or petri dish with 10 ml cetrimide and counting the released nuclei on a Coulter Counter model MHR (Coulter Electronics, Hialeah, FL) (data not shown) .
• Coulter Counter model MHR Coulter Electronics, Hialeah, FL
• Cytospin slides were prepared by centrifugation of 5,000-50,000 cells in cytospin funnels at 1,000 rpm for 5 minutes using a Shandon CytospinTM2 (Pittsburgh, PA) . The cells were then stained with Wright- Gie sa stain (Harleco, Gibbstown, NJ) for 30 seconds, followed by a phosphate buffer rinse for 1 minute. The slides were then evaluated for the presence of blast cells, promyelocytes, myelocytes, metamyelocytes, banded and segmented neutrophils, megakaryocytes, and promonocytes and monocytes.
• Colony assays were conducted as described in Example 1 above.
• the 0.8% methylcellulose colony assay medium was supplemented with 150 U/ml IL-3, 40 IL-6, 200 U/ml granulocyte-macrophage CSF (GM-CSF, R&D Systems) , 150 U/ml G-CSF and 10 U/ml erythropoietin (Epo, Amgen) .
• Fresh and cultured MNCs were plated between 1,000 and 9,000 cells/ml, while fresh and cultured CD34 * cells were plated between 500 and 4,500 cells/ml.
• CD33 antigens are partially degraded by chymopapain used to release CD34 * cells from the paramagnetic beads during CD34 + cell selection.
• M MNC is the multiplier for the MNC culture
• M-x ⁇ is the multiplier for the CD34 * cell culture
• X, ⁇ is the initial number of MNCs per culture
• X CD34 is the initial number of selected cells per CD34 * cell culture.
• %CD34 MNC is the %CD34 * cells in the MNCs (before selection)
• %CD34 CD34 is the % CD34 + cells in the CD34 + selected cells
• Y s is the yield of the selection process.
• PBMN Cells peripheral blood mononuclear cells.
• CD34+ Cells CD34+ selected cells.
• Bioreactor #1 and #2 perfusion culture using grooved bioreactor of the present invention.
• Control #1 and #2 static culture.
• BFU-E and CFU-Mix was observed in perfused and static cultures for only one of six experiments. In the other five experiments, small numbers of BFU-E were observed prior to day 10, and CFU-Mix were not observed beyond day 0.
• Perfusion MNC cultures supported the primitive LTC-IC better than static cultures over 15 days. Perfusion expanded the average number of LTC-IC to 123% of the input number at day 15 (183% at day 10) , although expansion was observed in only two of the experiments. In contrast, static cultures were only able to maintain LTC-IT at 74% of the input number on day 15 (89% at day 10) , with expansion observed in only one of six experiments. The fraction of cells giving rise to LTC-IC fell off continuously for both culture types from an average of 0.15% on day 0 to 0.02% or less on day 15.
• Perfusion cultured MNCs had a more primitive phenotype than the static cultured cells. Cells in perfusion maintained Table 4
• Table 4 Percent of cells from static and perfusion cultures of MNCs and CD34+ cells expressing CD34 and CD33.
• Perfusion CD34+ cell cultures supported LTC-IC better than static cultures over 15 days. Perfusion expanded the average number of LTC-IC to 135% of the input value on day 15, although expansion was observed in only two experiments. In contrast, static cultures were only able to maintain LTC-IC at 72% of the input number on day 15, with expansion observed in only one of six experiments. The fraction of cells giving rise to LTC-IC decreased continuously for both culture types from an average of 1.6% on day 0 to 0.02% or less on day 15. CD34+ cells in perfusion had similar phenotypes as the cells in static culture. Perfusion tended to maintain the CD34+ population longer, but the fraction of CD33+, CDllb- /CD15-, and CDllb/CD15+ cells were similar.
• CD34+ cells were primarily blast cells.
• the fraction of blast cells decreased rapidly, and the cultures contained predominantly granuiocytic and monocytic cells by day 10.
• CFU-GM, and LTC-IC contained primarily monocytic cells.
• perfused cultures were generally equivalent to or better than the static cultures for samples that grew well.
• perfusion culture provided at least limited expansion of samples that failed to grow in the static cultures.
• CDllb /CD15 were compared to CD33/CD34 and CDllb/CD15 .
• the CD34+ cells contained predominantly blast cells with some monocytic cells and very few gra nuisanceocytic cells, whereas the MNCs contained predominantly granuiocytic and monocytic cells with some blast cells.
• both cultures were predominantly granuiocytic with a large fraction of monocytic cells.
• the prevalence towards the granuiocytic lineage is greater than that indicated in Table 6 because: (1) mature granulocytes have short half-lives in culture even in the presence of G-CSF, (2) unidentifiable cells (about 10% of the total) are included with the monocytes, and (3) immature megakaryocytes are not distinguished from monocytes using Wright-Giemsa stain.
• perfusion cultures of PB MNCs and CD34+ cells appear to mature along the granuiocytic lineage in a similar fashion for the growth factor combination used.
• the fraction of cells giving rise to CFU-GM and LTC-IC in cultures initiated with MNCs and CD34 + cells was also similar after 10 days of perfusion culture.
• the fraction of cells giving rise to CFU-GM was 2.9% and 2.5% on day 10 for MNCs and CD34 * cells, respectively. This contrasts with 0.4% and 4.7% on day 0, respectively.
• the fraction of cells giving rise to LTC-IC was 0.10% and 0.043% on day 10 for MNCs and CD34 * cells, respectively. Again, this is in contrast to 0.15% and 1.6% on day 0, respectively.
• the total numbers of cells, CFU-GM and LTC-IC that could be obtained from perfusion culture of a peripheral blood sample cultured as MNCs are greater than those that could be obtained for the same sample selected and cultured as CD34 + cells (Table 7) .
• CD34+ 5 1,538 ⁇ 2,47 ⁇ t (62 - 6,455)
• ⁇ and T T Differences between MNCs and CD34+ cells (p ⁇ 0.05 and p ⁇ 0.01. respec t ively ) .
• the maximum number of total cells, CFU-GM and LTC-IC were obtained on days 15, 10-15 and 10-15, respectively.
• Perfusion culture seeded with MNCs would yield 1.5-, 2.6- and 2.1-fold more total cells, CFU-GM and LTC-IC, respectively, on day 15 than would selecting and culturing the CD34 + fraction, as determined using the culture performance, initial cell loading, and yield on the CD34 + cell selection obtained for each experiment (see Data Analysis) .
• the 19-fold maximum CFU-GM expansion obtained for MNC cultures compares favorably to the 3.8- to 16-fold expansion reported for peripheral blood MNCs (PBMNCs) ( Takaue et al., supra: McAlister, et al., supra) .
• PBMNCs peripheral blood MNCs
• the 11- to 18-fold maximum CFU-GM expansion for CD34 + cell cultures is lower than previously reported 57- to 190-fold expansions for PB CD34 + cells (Haylock DN, et al. , supra; Sato N, et al., supra; Brugger W. , et al., supra) .
• CD34 + cell cultures may also be due to differences in the feeding protocol (e.g., how depopulation is accounted for) , CD34 + cell selection methods and culture media used, and sample sources.
• PB samples were used from normal donors and cancer patients mobilized with chemotherapy and/or growth factor regimens. Peripheral blood from these sources can vary greatly in the fraction of primitive cells. For example, Brugger et al, indicate that only 0.2% of the CD34 * cells obtained from chemotherapy and G-CSF mobilized blood formed CFU-GM colonies.
• cultures inoculated with either MNCs or CD34 * cells produced cells that were remarkably similar after 10 days of culture. Changes observed in cell phenotype followed similar patterns of myeloid differentiation reported for cultures of bone marrow (Smith SL, et al., Exp Hematol 21:870-877, 1993) and cord blood (Terstappen LWMM, et al., Leukemia 6:1001-1010, 1992).
• CD34 * cells gain CD33 and lose CD34. The cells can further differentiate, with those maturing towards neutrophils acquiring CD15 followed by CDllb, while those maturing towards monocytes acquire CDllb and then CD15. This suggests that the CFU-GM present in expanded cell populations may be more mature than those present in uncultured cells. Infusion of large numbers of mature progenitor cells has the potential to decrease the extent and duration of cytopenias following transplantation.
• the MNC and CD34 + cell perfusion cultures can be compared directly in terms of the quantity of cells, CFU-GM and LTC-IC produced. After 15 days in perfusion culture, MNCs produced 1.5-,2.6- and 2.1-fold more total cells, CFU-GM and LTC-IC, respectively, than would the same sample selected and cultured as CD34 * cells. Even if the CD34 selection process was 100% efficient, production of CFU-GM would be 1.5-fold greater for MNCs than for CD34 * cells. This difference does not appear to be due to losses incurred during the selection process because when the yield on the CD34 selection is considered, 100( ⁇ 100)% of the CFU-GM and 70( ⁇ 30)% of the LTC-IC are recovered. While production of CFU-GM from MNC cultures may not exceed that from CD34 * cell cultures for all initial cell populations and culture conditions, our results clearly demonstrate that selection of CD34 + cells is not required in order to obtain extensive CFU-GM expansion.
• CD34 cell selection may still be desirable for reasons other than increasing cell expansion.
• tumor cells in breast Ross AA, et al., Blood 82:2605-2610, 1993
• small cell lung cancer Brugger W, et al., Blood 83:646-640, 1994
• CD34 * cells may still be required to provide additional purging. While stem cells per se are not required for reconstitution following myelosuppressive therapy, the decrease in LTC-IC numbers during mobilized blood culture may adversely affect long-term reconstitution following myeloablative therapy. Under these circumstances, it may be best to combine expanded cells (to provide large numbers of mature progenitors) with uncultured cells. In this regard, CD34 selection reduces the total volume for transplantation using uncultured cells and modulates graft vs. host disease in allotransplants. Finally, cultured CD34 * cells may increase the efficiency of transfection for gene therapy.
• perfusion culture a major advantage of perfusion culture is that those samples that performed very poorly in static culture exhibited at least limited (and in most cases normal) expansion in perfusion.
• perfusion cultures maintained LTC-IC numbers better than the static cultures, which is consistent with results for PB and CB MNCs on irradiated stroma (Koller,et al. , 1993, supra..
• Perfused bioreactors are superior to bag or flask cultures for progenitor cell expansion for transplantation because they maintain desired culture condition, minimize chances for contamination during feeding, are easier to scale up for clinical application, and facilitate compliance with current and expected Food and Drug Administration (FDA) regulations.
• FDA Food and Drug Administration
• CFR section 211 21 CFR 211
• GMP the set of regulations known commonly as GMP
• CFR 211.22 mandates the institution of a quality control unit, the head of which must be distinct from the transfusion center director.
• Increased FDA regulatory activity is anticipated for cellular therapies such as autolymphocyte therapy and bone marrow transplant. Ex vivo expansion of hematopoietic cells will most certainly be governed by 21 CFR 211.
• results obtained for total cell and CFU-GM expansion can be used to estimate the size of the initial mobilized blood sample and culture system required for therapeutic application of cultured hematopoietic cells.
• a therapeutic dose of 20 x 10 4 CFU-GM/kg body weight has been suggested for rapid engraftment of neutrophils using peripheral blood cells (Bender JG, et al., J Hematotherapy 1:329-341, 1992).
• An 80 kg individual would then require 16 x 10° CFU-GM.
• the culture system would have to accommodate at least 2 x 10 cultured MNCs. Since neither culture exhibited indications of limiting cell proliferation due to cell density, an estimate for the maximum cell density obtainable for the perfusion and static cultures can be found by dividing the maximum cell numbers obtained per culture by the culture surface area. The maximum obtained in perfusion was 48 x 10 cells on effectively 15 cm 2 culture area, or 3.2 x 10° cells/cm 2 assuming that the cells are only in the grooves.
• Example 3 Cord Blood Mononuclear Cells in Smooth versus Grooved Perfusion Chambers.
• CB cord blood
• Cytokine concentrations for all cultures were as in Example 1.
• Perfusion and static cultures were conducted as in Example 2.
• Culture medium was HLTM as in Example 1, containing 12.5% preselected lots of FBS and horse serum, respectively.
• Stroma-free CB MNC cultures, supplemented with IL-3, IL-6, G-CSF, and SCF, were conducted in both smooth perfusion culture chambers and the grooved bioreactor of the present invention. Control static cultures were conducted in petri dishes. No stroma was used in this series of experiments.
• Grooved perfusion culture using the grooved bioreactor of the present invention, no stroma.
• Control static culture in petri dish, no stroma.
• Perfusion cultures in the grooved bioreactor showed similar cell expansion as the cultures in the static cultures. However, perfusion cultures in the smooth chamber showed only one-half the cell expansion as perfusion cultures in the grooved bioreactor. Few, if any, cells were washed out of the grooved chamber as evidences by few, if any, cells being found in the cell trap after the grooved, but not the smooth, chamber. Viability was below 20% for the cells found in the cell trap.
• Perfusion cultures in the grooved chamber gave greater CFU- GM, BFU-E, and CFU-Mix expansion than static cultures in petri dishes.
• Maximum CFU-GM expansion of 22-, 10-, and 13-fold were obtained from cultures in the grooved chamber, smooth chamber, and petri dish respectively, on Day 10.
• maximum CFU-Mix expansion of 6.2-, 4.9, and 6.7-fold were obtained from cultures in the grooved chamber, smooth chamber, and petri dish, respectively, on day 5.
• the smooth chamber did not appear to preferentially retain specific cells over others as evidenced by the similar distribution of colony types and the fraction of cells giving rise to CFU-C in the cultures with grooved and smooth chambers.

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